Our long-term goal is to produce large amounts of recombinant human hemoglobin in Escherichia coli to allow commercial production of the protein for formulation of hemoglobin-based oxygen carriers which will be used for blood transfusions when human blood is not available. When hemoglobin is expressed in E. coli, sufficient heme must be available to be added to globin chains, or bacterial proteases degrade them before hemoglobin assembly occurs. Our hypothesis is that hemoglobin production can be increased by transforming E. coli with plasmids containing human hemoglobin genes and heme transport genes from a Gram-negative bacterium, and supplying heme in the media. Our work has indicated that this hypothesis is valid and that heme transport systems from Plesiomonas shigelloides and Shigella dysenteriae are best suited for this work. "One plasmid" systems were created which contain P. shigelloides heme transport genes or the S. dysenteriae heme receptor gene shuA on the same plasmid as the 1 and 2 hemoglobin genes. These single expression vectors were constructed to solve growth and hemoglobin expression problems that occur when the genes are on two separate plasmids. Heme transport genes also were moved into the chromosome of an E. coli strain transformed with plasmid encoded hemoglobin genes. These approaches resulted in strains that produced up to ten times more hemoglobin than those expressing only hemoglobin genes. Work also was performed to alter the promoter of shuA, which is normally activated by iron depletion, to facilitate shuA expression in iron rich medium where growth is more vigorous. The major goal of the work proposed in this application is to optimize the hemoglobin expression systems we developed in the past grant period. In addition, our heme transport strategy will be combined with a second approach in which genetically modified hemoglobin chains are constructed to enhance their stability and facilitate assembly into stable tetrameric hemoglobin in E. coli. The specific aims of this new proposal are: 1) Construct a one plasmid system containing human hemoglobin genes, the heme receptor gene shuA and other S. dysenteriae heme transport genes that will permit more heme to be transported into the cell than with shuA alone, and then test these strains for enhanced hemoglobin production. 2) Create additional versions of shuA with promoters that allow higher expression of shuA when the cells are grown in high iron media. 3) Move shuA or shuA versions with modified promoters into the chromosome of E. coli strains containing various plasmid encoded human hemoglobin genes and test these strains for enhanced hemoglobin production. 4) Construct one plasmid systems containing heme transport genes and human hemoglobin genes altered to encode more stable hemoglobins, and again test these strains for enhanced hemoglobin production. 5) Evaluate the "best" expression strains containing single plasmid or chromosomal systems in a bioreactor designed to mimic large scale production protocols. PUBLIC HEALTH RELEVANCE: The research is targeted at developing a blood substitute from hemoglobin produced in and harvested from bacteria. The blood substitute could be used when human blood is not available, and would not have the potential to carry human diseases such as AIDS or hepatitis. The goal of this research is to solve the production problems associated with making large amounts of hemoglobin in bacteria.